The art recognises the desirability of screening repertoires of cells or proteins to identify proteins of interest (POI) or cells expressing these. For example, the art comprises techniques to screen B-cells and repertoires of antibodies, in order to identify one or more cells that express antibodies displaying a desired characteristic (typically specific antigen binding). Screening is not confined to antibody screening, but also may be applicable to screening collections of other types of proteins for one or more desired characteristics.
In order to express protein repertoires, corresponding nucleotide sequences can be cloned into respective expression vectors and introduced (e.g., transfected) into host cells that can express the proteins. Commonly, molecular cloning is used to clone protein-encoding sequences from a repertoire into host cells. Molecular cloning has been applied to techniques of B-cell (lymphocyte) screening wherein B-cells are expanded by culturing, sorted into single cells (e.g., using a haemolytic plaque assay or fluorescent foci assay), antibody chain (or variable region) mRNA from the sorted cells is reverse transcribed and amplified using RT-PCR, amplified DNA undergoes molecular cloning to introduce antibody chain-encoding sequences into nucleic acid vectors and the vectors are then introduced into host cells for transient expression (where the vector is episomal) or for stable expression (by random integration into the host cell genome). See, for example, Proc Natl Acad Sci USA. 1996 Jul. 23; 93(15):7843-8, “A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities”, J S Babcook et al (known as the SLAM technique) and doi: 10.1016/j.jala.2009.05.004 Journal of Laboratory Automation October 2009 vol. 14 no. 5 303-307, “High-Throughput Screening for High Affinity Antibodies”, S Tickle et al (known as the UCB SLAM technique).
Molecular cloning involves PCR amplifying cDNA (produced by RT-PCR of mRNA of POIs) with primers carrying restriction enzyme cloning sites. This produces PCR products in which each POI nucleotide sequence is flanked by restriction sites. The PCR products are then digested with the appropriate restriction enzymes and subcloned into empty expression vectors which provide a promoter and polyA signal by ligation as well as carry a selection marker. The ligated products are then transformed into E. coli and clones are selected by the presence of the marker. It is necessary to confirm and select clones with correct sequence insertions by laborious restriction mapping and sequencing of individual clones. Each single correct clone expression vector is then individually purified from E. coli and transfected into a host cell (e.g., a CHO or HEK293 mammalian cell) for transient or stable expression.
Molecular cloning is, therefore, a laborious technique involving multiple steps and is not well suited to high-throughput.
Transient expression, such as from a linear expression cassette, is usually limited and stable expression is preferred for longer-term expression. For stable expression, typically the POI-encoding sequence of interest is inserted into the mammalian genome by the classical integration method of spontaneous integration of foreign DNA (i.e., random integration in the genome). This approach often leads to significant transcriptional variation (and thus unpredictability and inconsistent POI expression) as a result of differences in the transgene copy number and the site of integration (see Henikoff S (1992), “Position effect and related phenomena”, Curr Opin Genet Dev 2(6): 907-912; Martin D I, Whitelaw E (1996), “The variegating transgenes”, Boessays 18(11): 919-923; and Whitelaw E et al. (2001), “Epigenetic effects on transgene expression”, Methods Mol Biol. 158: 351-368). In addition, transgene fragments integrated in this way are often found to be inserted as concatemers which can result in gene inactivation by repeat-induced gene silencing (see Garrick D et al (1998), “Repeat-induced gene silencing in mammals”, Nat Genet 18(1): 56-59; and McBurney M W et al (2002), “Evidence of repeat-induced gene silencing in cultured Mammalian cells: inactivation of tandem repeats of transfected genes”, Exp Cell Res 274(1): 1-8). These problems hamper the production of repertoires of POIs and cell populations for expressing such POI repertoires.
Many technologies exist for the generation of monoclonal antibodies (mAbs) from human or transgenic animals carrying an antibody repertoire. Generally, mAbs are obtained from immortalization of B cells either by fusion (hybridoma technology) or transformation (virus transfection or oncogene transformation). These cell immortalization methods, however, are unsuitable for a comprehensive screening of large antibody repertories, because they are highly biased, inefficient and typically only sample a minute proportion of the available repertoire (typically less than 0.1% (immortalised cells/input cells) of a B-cell repertoire obtained from an immunised mouse, for example). The use of alternative B-cell screening methods that do not require immortalisation, therefore, is attractive, but techniques currently face the problems discussed above.